Tissue plasminogen activator is an enzyme widely used as a thrombolytic agent in the treatment of acute myocardial infarction. t-PA is secreted from endothelial cells as a single polypeptide chain that is subsequently cleaved (between Arg.sub.275 and Ile.sub.276) into two chains held together by a single disulfide bond (Rijken, D. C. et al., (1981) J. Biol. Chem. 256, 7035-7041. Both the single- and two-chain forms of the enzyme can bind to fibrin (Rijken, D. C. et al., (1982) J. Biol. Chem. 257, 2920-2925; Higgins, D. L. et al., (1987) Biochem. 26, 7786-7791), although the two-chain form is catalytically more active (Wallen, P. et al., (1982) Biochim. Biophys. Acta 719, 318-328; Ranby, M., (1982) Biochim Biophys. Acta 704, 461-469; Tate, K. M. et al., (1987) Biochem. 26, 338-343; and Petersen, L. C. et al., (1988) Biochim. Biophys. Acta 952, 245-254). In addition, both forms of the enzyme (Jorgensen, M. et al., (1987) Thromb. Haemostasis 58, 872-878) are inactivated by a fast-acting plasminogen activator inhibitor (type 1)(PAI-1), a member of the serpin family that is secreted from endothelial cells and forms a covalent bond with Ser.sub.478 of t-PA (Levin, E. G., (1983) Proc. Natl. Acad. Sci. USA 80, 6804-6808; and Sprengers, E. D. et al., (1987) Blood 69, 381-387).
Exogenously administered t-PA is capable of eliciting prompt thrombolysis in therapeutic doses that do not produce marked fibrinolysis in experimental animals with induced coronary artery thrombosis (Bergmann, S. R. et al., (1983) Science, 220, 118114 1183) and in patients with evolving myocardial infarction (Van de Werf, F. et al., (1984) New Eng. J. Med. 310, 609-613; Collen, D. et al., (1984) Circ. 70, 1012-1017; Van de Werf, F. et al., (1984) Circ. 69, 605-610; Thrombolysis in Myocardial Infarction Study Group, (1985) New Eng. J. Med. 312, 932-936; and European Cooperative Study Group, (1985) Lancet 1, 842-847). However, because the clearance of t-PA from the circulation is so rapid, continuous infusions have been required.
In vivo studies of t oPA clearance have been performed in a variety of species including mice (Fuchs, H. E. et al., (1985) Blood 65, 539-544); rats (Emeis, J. J. et al., (1985) Thromb. Haemost. 54, 661-664; Rijken, D. C. et al., (1986) Biochem. J. 238, 643-646; Kuiper, J. et al., (1988) J. Biol. Chem. 263, 18220-18224; Bakhit, C. et al., (1988) Fibrin. 2, 31-36; Krause, J. et al., (1990) Biochem. J. 267, 647-652); rabbits (Korninger, C. et al., (1981) Thromb. Haemostasis 46, 658-661; Bounameaux, H. et al., (1986) Blood 67, 1493-1497); dogs (Devries, S. R. et al., (1987) Fibrin. 1, 17-21; Yasuda, T. et al., (1988) J. Clin. Invest. 81, 1284-1291); and monkeys (Flameng, W. et al., (1985) J. Clin. Invest. 75, 84-90. These studies together with experiments in man (Garabedian, H. D. et al., (1986) Am. J. Cardiol. 58, 673-679; Verstraete, M. et al., (1986) Thromb. Haemostas. 56, 1-15) demonstrate the rapid removal of t-PA from the circulation, which varies from about tu.sub.1/2 =1 min in rats to t.sub.1/2 5 min in man.
The liver appears to be the major site of removal and catabolism of t-PA (Nilsson, T. et al., (1984) Scand. J. Haematol. 33, 49-53; Devries, S. R. et al., (1987) Fibrin. 1, 17-21; Korninger, C. et al., (1981) Thromb. Haemostasis 46, 658-661; Bounameaux, H. et al., (1986) Blood 67, 1493-1497; Beebe, D. P. et al., (1986) Thromb. Res. 43, 663-674; Nilsson, S. et al., (1985) Thromb. Res. 39, 511-521; Emeis, J. J. et al., (1985) Thromb. Haemost. 54, 661-664; Rijken, D. C. et al., (1986) Biochem. J. 238, 643-646; Fuchs, H. E. et al., (1985) Blood 65, 539-544; and Kuiper, J. et al., (1988) J. Biol. Chem. 263, 18220-18224). About 80% of exogenous t-PA delivered intravascularly rapidly accumulates in the liver and is subsequently degraded, with subsequent appearance of degradation products in plasma. These studies support a general clearance mechanism for t-PA in which uptake and degradation within the liver is followed by the release of the degradation products initially into the blood and subsequently into the urine.
Further, the half-life of circulating t-PA is markedly prolonged in animals subjected to hepatectomy (Bounameaux, H. et al., (1986) Blood 67, 1493-1497; and Nilsson, T. et al., (1984) Scand. J. Haematol. 33, 49-53). Neither the protease active site nor a specific glycosylation pattern appears to be a major determinant of hepatic recognition and degradation of t-PA in vivo (Fuchs, H. E. et al., (1985) Blood 65, 539-544), in perfused liver systems (Emeis, J. J. et al., (1985) Thromb. Haemost. 54, 661-664, or in isolated hepatocytes (Bakhit, C. et al., (1987) J. Biol. Chem. 262, 8716-8720. The clearance and catabolism of t-PA has been reviewed in detail (Krause, J. (1988) Fibrin. 2, 133-142). However, information is limited regarding the particular cell type responsible for clearance of t-PA.
After administration of fluorescent or radiolabelled t-PA to rats and subfractionation of the livers into parenchymal, endothelial, and Kupffer cells, it was found that parenchymal and endothelial cells constitute the major sites for hepatic uptake (Fuchs, H. E. et al., (1985) Blood 65, 539-544; Sprengers, E. D. et al., (1987) Blood 69, 381-387; Kuiper, J. et al., Fibrin, 2:28 (1988) and Bugelski, P. J. et al., (1989) Thromb. Res. 53, 287-303).
The uptake of t-PA into all liver cell types is inhibited by in vivo competition with unlabelled t-PA whereas glycoproteins such as mannan and ovalbumin inhibit the specific uptake of labelled t-PA in isolated liver endothelial cells. The endocytosis of t-PA is mediated, at least in part, by mannose receptors on endothelial cells (Einarsson, M. et al., (1985) Thromb. Haemost. 54, 270; and Kuiper, J. et al., (1988) Fibrin. 2, 28). Monensin, NH.sub.4 Cl, and cytochalasin B block the uptake and degradation of t-PA, indicating that the uptake is endocytotic and that the degradation is lysosomal. In hepatoma cell lines, representing parenchymal cells, t-PA clearance involves ligand binding, uptake, and degradation mediated by a high capacity, high-affinity specific receptor system (Owensby, D. A. et al., (1988) J. Biol. Chem. 263, 10587-10594). Subfractionation of rat liver parenchymal, endothelial, and Kupffer cells 5 minutes after .sup.125 I-t-PA injection revealed that liver parenchymal cells are responsible for about 55% of the cleared .sup.125 I-t-PA, endothelial cells for about 40%, and Kupffer cells for about 5% (Kuiper, J. et al., (1988) J. Biol. Chem. 263, 18220-18224; Rijken, D.C. et al., (1990) Thromb. Res. Suppl. X, 63-71).
Two distinct mechanisms for t-PA catabolism by hepatoma cells have been shown. t-PA complexed to PAI-1 is recognized by a PAI-1 dependent receptor on the cell surface of human hepatoma HepG2 cells (Schwartz, A. L. et al., (1981) J. Biol. Chem. 256, 8878-8881; Owensby, D. A. et al., (1988) J. Biol. Chem. 263, 10587-10594; Morton, P. A. et al., (1989) J. Biol. Chem. 264, 7228-7235; Bu, G. et al., (1992) J. Biol. Chem. 267, 15595-15602). t-PA in the absence of bioactive PAI-1 has been found to bind to PAI-1 independent receptors which mediate binding and endocytosis of t-PA on rat hepatoma MH.sub.1 C.sub.1 cells (Bu, G. et al., (1992) J. Biol. Chem. 267, 15595-15602) and on rat Novikoff hepatoma cells (Nguyen, G. et al., (1992) J. Biol. Chem. 267, 6249-6256). Although this PAI-1 independent t-PA clearance system has not been reported on human hepatocytes, the rapid clearance of intravenously injected t-PA, normally at a level far exceeding the available PAI-1, suggests the existence of a PAI-1 independent t-PA clearance system.
At present, t-PA is administered clinically in the form of an initial bolus that is followed by sustained infusion. The total amount of enzyme administered during a standard 3 hour treatment is 50-100 mg. Such large amounts are required for two reasons: first, to counterbalance the effects of the rapid clearance of t-PA from the circulation, and second, to overcome the effects of high concentrations of fast-acting inhibitors of the enzyme that are present in plasma and platelets. When high doses are used in an effort to increase the rate of clot lysis or to lyse refractory clots, there is a risk of systemic fibrinolysis which affects the body's capacity to stop bleeding and hemorrhage.
Reducing the rate of t-PA clearance from the circulation following administration would provide a significant advantage in clinical use. This would allow t-PA to be administered in much smaller doses than are currently required, thereby reducing, e.g., the risk of systemic fibrinolysis and hemorrhage. Accordingly, an objective of the invention is to provide methods and compositions for the inhibition of hepatic clearance of t-PA. Another objective of the invention is to provide a method and composition for the treatment of thrombolytic diseases which allows t-PA to be administered in much smaller doses than currently required. These and other objectives and features of the invention will be apparent from the following description.